Distinct Roles for Neutrophils and Dendritic Cells
in Inflammation and Autoimmunity in motheaten Mice
Clare L. Abram,1Gray L. Roberge,1Lily I. Pao,2,3Benjamin G. Neel,2,4,* and Clifford A. Lowell1,*
1Department of Laboratory Medicineandthe Programin Immunology, University of California,San Francisco, San Francisco, CA 94143, USA
MA 02215, USA
3Present address: Five Prime Therapeutics, Inc., South San Francisco, CA 94080, USA
4Present address: Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health
Network and Department of Medical Biophysics, University of Toronto, Toronto, Ontario MG5 1L7, Canada
*Correspondence: firstname.lastname@example.org (B.G.N.), email@example.com (C.A.L.)
for complex autoimmune and inflammatory disease.
Null mutations in Ptpn6, which encodes the nonre-
ceptor protein-tyrosine phosphatase Shp1, cause
the motheaten phenotype. However, Shp1 regulates
multiple signaling pathways in different hematopoi-
etic cell types, so the cellular and molecular mecha-
nism of autoimmunity and inflammation in the
motheaten mouse has remained unclear. By using
floxed Ptpn6 mice, we dissected the contribution of
innate immune cells to the motheaten phenotype.
Ptpn6 deletion in neutrophils resulted in cutaneous
inflammation, but not autoimmunity, providing an
animal model of human neutrophilic dermatoses.
By contrast, dendritic cell deletion caused severe
autoimmunity, without inflammation. Genetic and
biochemical analysis showed that inflammation was
caused by enhanced neutrophil integrin signaling
through Src-family and Syk kinases, whereas auto-
immunity resulted from exaggerated MyD88-depen-
dent signaling in dendritic cells. Our data demon-
strate that disruption of distinct Shp1-regulated
pathways in different cell types combine to cause
Shultz in 1975 as a spontaneous autosomal recessive mutation
leading to immune deficiency, widespread inflammation, and
death at 2 to 4 weeks of age (Green and Shultz, 1975). Later,
a related mutant strain was discovered (motheaten viable, or
mev/mev) in which the homozygous mice display a similar,
though less severe, phenotype and survive for several more
(9–12) weeks (Shultz et al., 1984). Both lines of mice exhibit
chronic inflammation of the skin (leading to fur loss, hence the
name ‘‘motheaten’’), accompanied by the production of autoan-
tibodies and immune complex deposition in tissues. me/me and
mev/mevmice also develop a lethal pneumonitis characterized
by neutrophil and macrophage accumulation in the lungs
(Jiao et al., 1997). Gene-mapping studies show that distinct
mutations in Ptpn6, which encodes the protein-tyrosine phos-
phatase Shp1, cause the me/me and mev/mevphenotypes.
The Ptpn6me/memutation results in complete loss of Shp1
protein, whereas Ptpn6me-v/me-vmice express wild-type (WT)
amounts of two mutant forms of Shp1 with greatly reduced cata-
lytic activity (Shultz et al., 1997). Recently, two strains of mice
with new Ptpn6 point mutations were reported, Ptpn6spin/spin
and Ptpn6meB2/meB2, both of which have milder phenotypes
than seen in Ptpn6me-v/me-vmice (Croker et al., 2008; Nestero-
vitch et al., 2011a). The motheaten phenotype has long served
as a paradigm for a complex autoimmune and inflammatory
disease; therefore, elucidating its pathogenesis is of general
Substantial effort has been devoted to investigating the func-
tion of Shp1 in the immune system (Pao et al., 2007a; Tsui et al.,
2006). Nevertheless, its detailed role in WT mice, and an under-
standing of how Ptpn6 mutations cause autoimmunity and
inflammation, remains unclear. All hematopoietic cells express
Shp1, and their complicated interactions have made it difficult
to dissect the relative contributions of different cell types to mo-
theaten disease. Transplantation experiments indicate that the
motheaten phenotype is due predominantly to bone-marrow-
derived cells. Moreover, pretreatment of motheaten mice with
anti-CD11b prevents the development of skin inflammation,
pneumonitis, splenomegaly, and defective T cell function (Koo
et al., 1993). These data indicate that myeloid cells not only
cause the inflammatory disease but also influence the develop-
ment of autoimmunity in motheaten mice. Consistent with this
conclusion, analysis of Ptpn6me-v/me-vmice on a Rag1-deficient
background shows that B and T lymphocytes are dispensable
for the inflammatory disease (Yu et al., 1996).
In addition to acting in several hematological cell types,
Shp1 is implicated in the regulation of multiple signaling path-
kinases, G protein coupled receptors, Toll-like receptors (TLRs),
and cytokine receptors (Neel et al., 2003; Pao et al., 2007a;
Zhang et al., 2000). Much previous work has focused on study-
ing cells isolated from mice containing Ptpn6 mutations. For
example, neutrophils from Ptpn6me/meand Ptpn6me-v/me-vmice
are hyperadhesive and show defective chemotaxis, perhaps
Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc. 489
Figure 1. Disease Phenotype in Ptpn6fl/flS100a8-cre and Ptpn6fl/flItgax-cre Mice
(A and C) Mice of the indicated genotypes were monitored every two (Ptpn6fl/flS100a8-cre) or four (Ptpn6fl/flItgax-cre) weeks for paw inflammation or lymph-
adenopathy, respectively. The presence of these phenotypes was scored, and the percentage of ‘‘disease-free’’ mice at each time point was graphed using
Prism, ***p < 0.001.
(legend continued on next page)
Deconvoluting the Phenotype of motheaten Mice
490 Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc.
reflecting an inability to de-adhere due to exaggerated
integrin signaling (Kruger et al., 2000). Macrophages from
Ptpn6me-v/me-vmice also are hyperadhesive to integrin ligands
and are hyperresponsive to colony-stimulating factor-1 (CSF-1)
and granulocyte-macrophage colony stimulating factor-1 (GM-
CSF) (Chen et al., 1996; Jiao et al., 1997; Roach et al., 1998).
In dendritic cells (DCs), Shp1 reportedly regulates multiple TLR
and cytokine signaling responses (An et al., 2008; Ramachan-
dran et al., 2011). In general, myeloid cells lacking Shp1 are
hyperresponsive to multiple stimuli, but it is unclear which
stimuli, acting in which cell types, are critical to the develop-
ment of the motheaten phenotype. Furthermore, alterations in
immune cell function might arise as a consequence of indirect
effects caused by the inflammatory milieu in these animals,
rather than from cell-intrinsic abnormalities caused by loss of
The development of a conditional (floxed) Ptpn6 allele (Pao
et al., 2007b) has facilitated investigation of the roles of specific
cell types in the pathogenesis of the motheaten phenotype. Mice
lacking Shp1 in B cells show perturbed B cell development,
specifically an expansion of B1a cells at the expense of B2 cells,
and aberrant B cell receptor signaling in B1a cells. These mice
develop autoimmune disease, but they do not develop the
inflammatory skin or lung disease characteristic of motheaten
mice (Pao et al., 2007b). Mice lacking Shp1 in T cells develop
neither autoimmunity nor inflammatory disease (Fowler et al.,
2010). In this study, we investigated the contribution of specific
myeloid cell subsets to the motheaten phenotype by crossing
Ptpn6fl/flmice with mice that express Cre in distinct lineages of
myeloid cells. Our results provide insights into the pathogenesis
of the motheaten phenotype and provide a molecular framework
for understanding the complex interactions between immune
cells that drive autoimmune and inflammatory diseases.
Specificity of Myeloid Cre Deletion
We set out to determine the contribution of Shp1-deficient
myeloid cells to the motheaten phenotype by crossing Ptpn6fl/fl
mice to several myeloid Cre lines. To assess the efficiency
and specificity of deletion, we also crossed each Cre line to
ROSA26-lox-stop-lox-EYFP reporter (ROSA-YFP) mice, and
then quantified the percentage of YFP+cells by flow cytometry.
In Vav1-cre:ROSA-YFP mice, all hematopoietic cells analyzed
were 98%–100% YFP+(data not shown). S100a8 (also known
as MRP8)-cre:ROSA-YFP mice showed specific deletion in
neutrophils, with little or no YFP expression in macrophages,
monocytes, NK cells, mast cells, basophils, or eosinophils
(data not shown). As the S100a8-cre transgene contains an
IRES-GFP cassette, we also followed Cre expression by flow
cytometry; these data confirmed that in Ptpn6fl/flS100a8-cre
and Ptpn6+/+S100a8-cre mice, the Cre transgene was ex-
available online). Furthermore, intracellular staining by flow
cytometry revealed an absence of Shp1 in neutrophils from
Ptpn6fl/flS100a8-cre and Ptpn6fl/flVav1-cre mice (Figure S1B).
Ptpn6+/+and Ptpn6fl/flS100a8-cre mice showed equivalent
Shp1 expression in all other hematopoietic cell types (data
not shown). Biochemical analysis of neutrophils isolated from
bone marrow confirmed that Ptpn6fl/flS100a8-cre neutrophils
expressed ?20% of the WT amount of Shp1 (Figure S1C). By
contrast, bone-marrow-derived macrophages from Ptpn6fl/fl
S100a8-cre mice expressed normal amounts of Shp1 and
showed normal basal tyrosine phosphorylation, unlike Shp1-
deficient macrophages from Ptpn6fl/flVav1-cre mice (Fig-
ure S1D). We conclude that Ptpn6fl/flS100a8-cre mice show
neutrophil-specific deletion of Shp1.
To achieve specific loss of Shp1 in DCs, we crossed Ptpn6fl/fl
mice to Itgax (also known as Cd11c)-cre mice. Itgax-cre:ROSA-
YFP mice showed efficient deletion in DCs and alveolar macro-
phages (data not shown). Biochemical analysis showed that
splenic CD11chicells from Ptpn6fl/flItgax-cre mice expressed
Shp1 at ?18% of WT amounts (Figure S1E). Likewise, splenic
CD11chiMHCIIhiconventional DCs (cDCs) and CD11cintB220+
Ly6c+plasmacytoid DCs (pDCs) from Ptpn6fl/flItgax-cre and
Ptpn6fl/flVav1-cre mice had reduced Shp1 amounts (Figure S1F).
B and T lymphocytes from Ptpn6fl/flItgax-cre mice had normal
amounts of Shp1 by immunoblotting and flow cytometry (Fig-
ure S1E;data notshown). Thus, theItgax-cre transgene afforded
efficient and specific Ptpn6 deletion in cDCs and pDCs.
Mice Lacking Shp1 Selectively in Neutrophils or DCs
Exhibit Distinct Phenotypes
We found that we could separate the inflammatory and autoim-
mune phenotypes of motheaten mice by limiting myeloid dele-
tion of Ptpn6 to neutrophils or DCs, respectively. Ptpn6fl/fl
S100a8-cre mice developed spontaneous paw inflammation
beginning at 8–12 weeks of age, which was 85% penetrant on
a mixed 129Ola;C57BL/6 background and fully penetrant on
the C57BL/6 background (Figure 1A). Histological analysis of
the inflamed paws showed thickened dermal and epidermal
layers and bone-marrow hypercellularity (Figure 1B). The dermal
inflammation peaked at 8–10 weeks and then partially, but never
completely, resolved, suggesting that the mice were able to
of the dermal inflammation in the paws of Ptpn6fl/flS100a8-cre
mice matched that seen in Ptpn6fl/flVav1-cre and Ptpn6me-v/me-v
mice (data not shown).
(B)H&E-stained sectionsof normaland inflamed pawsfromWT S100a8-cre orPtpn6fl/flS100a8-cre mice.Original magnification was310(top)and 340(bottom).
Thickened epidermal and dermal layers are indicated by blue and black arrowheads, respectively; bone-marrow hypercellularity is indicated by the asterisk.
(D) Lymph node weights, relative to total mouse weight, for the indicated genotypes are shown; each circle represents a single mouse. ***p < 0.001. Photograph
shows representative lymph nodes from WT Itgax-cre or Ptpn6fl/flItgax-cre mice. Scale bars represent 5 mm.
(E) Spleen weights, relative to total mouse weight, for the indicated genotypes are shown; each circle represents a single mouse. *p < 0.05, ***p < 0.001.
Photograph shows representative spleens from WT Itgax-cre or Ptpn6fl/flItgax-cre mice. Scale bars represent 5 mm.
(F) Detection of anti-nuclear antibodies in serum by immunostaining of HEp-2 cells.
(G) Detection of anti-DNA IgG antibodies in serum by ELISA (error bars indicate SEM). **p < 0.01.
(H) H&E-stained sections of kidneys, showing glomerulonephritis in Ptpn6fl/flItgax-cre mice. Original magnification was 320.
Data were pooled from six independent experiments. See also Figure S1.
Deconvoluting the Phenotype of motheaten Mice
Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc. 491
By contrast, Ptpn6fl/flItgax-cre mice did not exhibit paw
inflammation, but instead, developed lymphadenopathy, which
began at ?15 weeks of age (Figure 1C) and became fully
penetrant by 40 weeks (Figure 1D). All Ptpn6fl/flItgax-cre mice
had splenomegaly at 40 weeks of age (Figure 1E), and these
mice also developed anti-nuclear antibodies (Figures 1F and
1G) and glomerulonephritis (Figure 1H). Ptpn6fl/+Itgax-cre mice
showed an intermediate degree of splenomegaly, but not
lymphadenopathy, at 40 weeks of age (Figures 1D and 1E) and
also developed anti-nuclear antibodies (data not shown). Thus,
even heterozygous deficiency of Shp1 in DCs was sufficient to
cause some degree of autoimmunity. Notably, at no time did
Ptpn6fl/flS100a8-cre mice show any evidence of autoimmune
Neither neutrophil- nor DC-specific deletion of Ptpn6 caused
the lethal inflammatory lung disease observed in motheaten
and motheaten viable mice. Although blinded histological anal-
ysis of lung sections showed that some Ptpn6fl/flS100a8-cre
and Ptpn6fl/flItgax-cre animals had diffuse or patchy inflamma-
tory cell infiltrates in their lungs, these were mild (Figure S1G).
Even combined deficiency of Shp1 in neutrophils, alveolar
macrophages and DCs (i.e., in Ptpn6fl/flS100a8-cre;Itgax-cre
mice) failed to induce diffuse inflammatory lung disease. By
contrast, Ptpn6fl/flVav1-cre mice exhibited the same diffuse
lung inflammation seen in motheaten strains and died within
weeks of birth.
The increase in splenocyte number was modest in Ptpn6fl/fl
S100a8-cre mice, compared with that seen in Ptpn6fl/flItgax-
cre animals (Figure 2A). By 15–20 weeks of age, Ptpn6fl/fl
S100a8-cre mice had moderately increased numbers of splenic
and bone-marrow neutrophils compared with controls (Fig-
ure 2B) and no alterations in splenic T or B cell numbers or acti-
vation status (Figure 2E; data not shown). By contrast, spleens
from Ptpn6fl/flItgax-cre mice showed disrupted architecture,
with a loss of germinal centers and blurring of the border
between the white and red pulp (Figure 2C). Splenomegaly in
these mice was caused by increased numbers of both myeloid
and lymphoid cells (Figures 2D and 2E). The numbers of cDCs
and pDCs were increased, and both showed significant activa-
tion, as indicated by upregulation of CD86 and major histocom-
patibility complex class II glycoproteins (MHCII), respectively
(Figures 2F and 2G). Ptpn6fl/flItgax-cre mice also showed
dramatic increases in CD4+T lymphocytes and in several B
lation of the early activation marker CD69, and there also was an
expansion of the effector and memory CD44hiCD62LloT cell
compartment (Figures S2B and S2C). Follicular B cells showed
upregulation of MHCII and CD86 (Figure S2E; data not shown).
We also analyzed a group of Ptpn6fl/flItgax-cre mice at
8–11 weeks of age, before signs of autoimmunity were apparent.
Spleen cell numbers were not increased significantly at this
time, but DC activation was present, indicating that the latter
precedes the development of autoimmune disease (Figure S2F).
Analysis of peripheral blood samples from Ptpn6fl/flItgax-cre
mice revealed a dramatic expansion of the CD11b+compart-
ment, compared with controls or Ptpn6fl/flS100a8-cre mice,
that was concomitant with a gradual increase in anti-nuclear
and anti-dsDNA antibody titers (Figures S2G–S2K). We also
measured cytokine concentrations in the sera from both groups
of mice using multiplex bead analysis. Not surprisingly, by
30 weeks of age, Ptpn6fl/flItgax-cre mice showed elevated
concentrations of all cytokines examined, reflecting the general
cell expansion and immune cell activation in these animals (Fig-
ure S2L). By contrast, only G-CSF was increased in the sera of
16- to 20-week-old Ptpn6fl/flS100a8-cre mice, compared with
controls, which likely accounts for the increased number of
immature granulocytes in the spleens of these animals (Figures
S1A and S2M). Sera from Ptpn6fl/flItgax-cre mice at the same
age showed elevated concentrations of cytokines, similar to
those observed at 30 weeks of age (data not shown). At earlier
times, which coincided with peak paw inflammation, Ptpn6fl/fl
S100a8-cre mice also had higher concentrations of keratino-
cyte-derived cytokine (KC), IL-6, and macrophage inflammatory
protein–1a (MIP-1a) (Figure S2N).
Inflammation in Motheaten Mice Is Caused by Abnormal
Integrin Signaling in Neutrophils
Previous work suggests that increased IL-1b signaling in
neutrophils is a major contributor to inflammation in the mo-
theaten mouse (Croker et al., 2008). Because IL-1b signaling
is mediated through the MyD88 adaptor protein, we tested
the role of this pathway by crossing motheaten viable mice
with Myd88?/?animals but, surprisingly, failed to obtain
any live progeny. We also failed to generate any live
Ptpn6fl/flMyd88fl/flVav1-cre mice, although we did obtain ex-
pected numbers of day 17 embryos with that genotype (Fig-
ure S3A). These data indicate that combined Shp1 and
MyD88 deficiency causes some uncharacterized defect in late
embryonic development. However, Ptpn6fl/flMyd88fl/flS100a8-
cre mice (i.e., mice with Shp1 and MyD88 deficiency restricted
to neutrophils) exhibited the same inflammatory disease as
Ptpn6fl/flS100a8-cre mice (Figure 3A). Further analysis of
double-mutant mice showed that neutrophil-specific deletion
of Myd88 did not reverse the increase in spleen size or spleno-
cyte number seen in Ptpn6fl/flS100a8-cre animals (Figures S3B
and S3C). There also was no significant decrease in the number
of splenic neutrophils compared to Ptpn6fl/flS100a8-cre mice
(Figure S3E). Deletion of Myd88 in neutrophils did, however,
reduce the increase in bone-marrow cellularity found in
Ptpn6fl/flS100a8-cre mice (Figures S3D and S3E). These data
indicate that the inflammation caused by deficiency of Shp1
in neutrophils does not result from excessive signaling through
MyD88-dependent pathways. Consistent with this notion, there
was no difference in lipopolysaccharide (LPS)- or Pam3CSK4-
evoked p38 MAPK phosphorylation in Shp1-deficient neutro-
phils (Figure S3F). Similarly, fMLF- or C5a-stimulated Erk phos-
phorylation, G-CSF-stimulated Stat3 phosphorylation and GM-
CSF-stimulated Stat5 phosphorylation were unaffected or
slightly hyporesponsive (Figure S3G). These results suggest
that Shp1 is not a major regulator of TLR, G protein-coupled,
or cytokine receptor responses in neutrophils and that these
rin stimulation (Kruger et al., 2000), so we asked whether loss of
Shp1 function downstream of integrin signaling might contribute
to inflammatory dermatitis. Indeed, compared with controls,
neutrophils from Ptpn6fl/flS100a8-cre mice produced more
Deconvoluting the Phenotype of motheaten Mice
492 Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc.
superoxide when plated on pRGD (which engages b1 and b2 in-
tegrins on these cells) in the presence or absence of tumor
necrosis factor-a (TNF-a) (Figure 3B). We also observed
enhanced activation-specific phosphorylation of Erk (Figure 3C)
and Src-family kinases (SFKs; Figure 3D) in neutrophils from
Ptpn6fl/flS100a8-cre mice plated on integrin ligands. No differ-
ence in the expression of SFKs was observed in neutrophils
from these mice (Figure S3H). These data suggested that Shp1
might regulate integrin signaling in neutrophils by influencing
Previous work from our group has shown that Syk is required
for neutrophil integrin signaling (Mo ´csai et al., 2002). We hypoth-
esized that deletion of Syk in neutrophils would reverse the
inflammatory disease in found Ptpn6fl/flS100a8-cre mice. We
tested this by generating Ptpn6fl/flSykfl/flS100a8-cre mice, which
lack both Shp1 and Syk specifically in neutrophils. Remarkably,
these mice were completely free of paw inflammation (Fig-
ure 4A) and showed normal numbers of spleen and bone
marrow neutrophils (Figures 4B–4E). Neutrophils from these
mice showed reduced superoxide production (Figure 4F) and
compared to neutrophils from Ptpn6fl/flS100a8-cre mice. These
data indicate that loss of Syk in neutrophils alone prevents
the motheaten inflammatory phenotype by reducing signaling
Figure 2. Lymphoid and DC Expansion and Activation in Ptpn6fl/flItgax-cre Mice
Cells were harvested from spleens and bone marrow, counted by using a Nucleocounter, stained with fluorescently conjugated antibodies, and analyzed by flow
(A) Total numbers of splenocytes in Ptpn6fl/flS100a8-cre and Ptpn6fl/flItgax-cre mice, compared with controls at 15–20 weeks of age; each circle represents one
(B) Total numbers of CD11b+Gr1hineutrophils in the spleen and bone marrow of Ptpn6fl/flS100a8-cre mice.
(C) H&E-stained sections of spleens from 40-week-old Ptpn6+/+Itgax-cre and Ptpn6fl/flItgax-cre mice. Original magnification was 34 (top) and 310 (bottom).
(D) Total numbers of splenic neutrophils and monocyte and macrophage subsets in Ptpn6fl/flItgax-cre mice.
(E) Total numbers of splenic T and B cells in Ptpn6fl/flS100a8-cre and Ptpn6fl/flItgax-cre mice, compared with controls.
(F and G) Numbers of total splenic cDCs (CD11chiMHCIIhi) or pDCs (CD11cintB220+Ly6c+) and expression of activation markers in Ptpn6fl/flItgax-cre mice.
Each bar represents an n of 5–10 mice at 15–20 weeks of age per group (error bars indicate SEM). *p < 0.05, **p < 0.01, ***p < 0.001. Data were pooled from six
independent experiments. See also Figure S2.
Deconvoluting the Phenotype of motheaten Mice
Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc. 493
downstream ofintegrins. SFKs alsoarecritical forthe initiation of
integrin signaling in neutrophils (Pereira and Lowell, 2003; Suen
et al., 1999), and we observed reversal of motheaten inflamma-
tory disease in Ptpn6me-v/me-vmice that also lacked the SFKs
Hck and Fgr or Hck, Fgr, and Lyn (data not shown). We conclude
that the inflammatory phenotype in motheaten mice primarily
results from dysregulated neutrophil integrin signal transduction
due to increased SFK and Syk activity.
Autoimmunity in Motheaten Mice Results from
Enhanced TLR Signaling in DCs
by the absence of Shp1 in neutrophils, MyD88 deficiency largely
prevented the autoimmune disease caused by lack of Shp1 in
DCs. Ptpn6fl/flMyd88fl/flItgax-cre mice had reduced spleen and
lymph node weights compared with Ptpn6fl/flItgax-cre mice
(Figures 5A and 5B), and sera from these mice lacked anti-
nuclear (Figure 5C) or anti-DNA (Figure 5D) antibodies. These
mice also showed significant reductions in splenic plasma cell
numbers andT cellactivation, and astriking reduction in myeloid
expansion (Figures 5E–5G). The number and activation status
of both cDCs and pDCs in these mice were reduced (Fig-
ure 5H). Immunoblot analysis confirmed that the amounts of
Shp1 and MyD88 protein were reduced in DCs from spleens of
Ptpn6fl/flMyd88fl/flItgax-cre mice (Figure S4). These data indicate
that loss of MyD88 in DCs prevents motheaten autoimmune
disease by blocking autoantibody production and preventing
T cell activation and myeloid expansion.
Previous studies have reported that Shp1 is a negative regu-
lator of TLR signaling in macrophages and DCs (Hardin et al.,
2006; Ramachandran et al., 2011). Indeed, BMDCs from
Ptpn6fl/flVav1-cre mice showed enhanced phosphorylation of
Erk and NF-kB (p65) and increased TNF-a production following
LPS stimulation (Figure 6A; Figures S5C and S5D). We also
observedincreased total, aswellasSyk andSFK,tyrosine phos-
phorylation in Shp1-deficient DCs (Figure S5A). The Shp1
substrate Sirpa also was hyperphosphorylated in Shp1-deficient
BMDCs (Figure S5B). Importantly, these abnormalities were
reversed by reexpression of Shp1, indicating that they reflect
effects of Shp1 deficiency on signaling per se, rather than poten-
tial indirect effects of the floxed Ptpn6 allele on adjacent genes
or DC development (Figures S5B–S5D). Because our genetic
evidence suggested that dysregulation of MyD88-mediated
pathways in DCs underlies the autoimmune disease caused by
Shp1 deficiency in these cells, we examined TLR signaling
responses in cells from Ptpn6fl/flItgax-cre mice. Splenic DCs
lacking Shp1 showed dramatically enhanced Erk activation
in response to LPS or CpG stimulation ex vivo (Figure 6B).
Hyperactivation of the Erk pathway in Shp1-deficient DCs
correlated with an increase in TNF-a production induced in
these cells in response to either TLR agonist (Figure 6C). By
contrast, GM-CSF-stimulated Stat5 phosphorylation and IL-
6-stimulated Stat3 phosphorylation were unaffected, or even
mildly hyporesponsive (Figures S5E and S5F). Moreover, LPS
signaling responses were normalized in splenic DCs from
Ptpn6fl/flMyd88fl/flItgax-cre mice (Figures 7A and 7B). We
Figure 3. Neutrophils from Ptpn6fl/flS100a8-cre Mice Show Hyperactive Integrin Signaling
(A) Mice of the indicated genotypes were monitored weekly for paw inflammation, and the percentage remaining ‘‘disease-free’’ was graphed using Prism.
(B) Neutrophils isolated from mice of the indicated genotypes were plated onto the integrin ligand poly-RGD (pRGD) in the presence or absence of TNF-a, and
superoxide release was measured (error bars indicate SEM).
(C and D) Neutrophils from Ptpn6fl/flor Ptpn6fl/flS100a8-cre mice were kept in suspension or plated onto pRGD-coated plates for the indicated times, lysed, and
analyzed by SDS-PAGE, followed by immunoblotting with the indicated antibodies. Data shown are representative of at least three individual experiments. See
also Figure S3.
Deconvoluting the Phenotype of motheaten Mice
494 Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc.
Figure 4. Neutrophil-Specific Syk Deficiency Reverses the Inflammatory Disease in Ptpn6fl/flS100a8-cre Mice
(A) Mice of the indicated genotypes (WT C57BL/6, n = 14; Ptpn6fl/flS100a8-cre, n = 17; Ptpn6fl/flSykfl/flS100a8-cre, n = 9) were monitored for paw inflammation,
and the percentage of disease-free mice at each time point was graphed using Prism, ***p < 0.001.
(B) Spleen weights, relative to total mouse weight, for the indicated genotypes; each circle represents a single mouse, ***p < 0.001.
(C–E) Spleen and bone-marrow cells from mice of the indicated genotypes were harvested, counted by using a nucleocounter, then stained with fluorescently
conjugated antibodies and analyzed by flow cytometry. Each circle represents a single mouse. Bars represent the average of 9–17 mice per group (error bars
indicate SEM). *p < 0.05, **p < 0.01, ***p < 0.001.
(F) Neutrophils isolated from mice of the indicated genotypes were plated onto the integrin ligand pRGD in the presence or absence of TNF-a, and superoxide
release was measured (error bars indicate SEM).
(G) Neutrophils from WT C57BL/6, Ptpn6fl/flS100a8-cre, and Ptpn6fl/flSykfl/flS100a8-cre mice were kept in suspension or plated onto pRGD-coated plates for
Deconvoluting the Phenotype of motheaten Mice
Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc. 495
conclude that Shp1-mediated regulation of TLR signaling path-
ways in DCs is an important factor in the development of autoim-
mune disease in motheaten mice.
We have determined the respective contributions of neutrophils
and DCs to the complex disease phenotype in motheaten mice.
We found that deficiency of Shp1 in neutrophils is sufficient
to cause spontaneous skin inflammation, whereas Shp1-defi-
ciency in DCs leads to severe autoimmunity. The autoimmune
phenotype observed upon deletion of Ptpn6 in DCs provides
one of the first examples that dysregulation of this single innate
immune cell type can cause autoimmune disease (see also Ka-
neko et al., 2012). We also identified key, and distinct, signaling
pathways important for the Shp1-deficient phenotype in each of
these cell types. Shp1 has been implicated in the regulation of
multiple signaling cascades, so one might expect that Shp1
deficiency would cause global activation of many cellular path-
ways. Surprisingly, we found that this was not the case; instead,
inflammation and autoimmunity in this model result from aber-
rant activation of integrin signaling in neutrophils and TLR
signaling in DCs.
Several lines of evidence indicate that the spontaneous skin
inflammation observed upon specific deletion of Ptpn6 in
neutrophils is due to hyperactive integrin signaling. For
Figure 5. Deletion of Myd88 in DCs Reverses the Autoimmunity in Ptpn6fl/flItgax-cre Mice
(A and B)Spleen and lymph node weights were obtained from the indicated groups of mice at 40 weeksof age and expressed as a fraction of total mouseweight.
Each circle represents a single mouse, ***p < 0.001.
(C) Detection of anti-nuclear antibodies in serum by immunostaining of HEp-2 cells.
(D) Detection of anti-DNA IgG antibodies in serum by ELISA (error bars indicate SEM) *p < 0.05, **p < 0.01.
(E–H) Cells were harvested from the spleens of mice with the indicated genotypes, counted by using a Nucleocounter, stained with fluorescently conjugated
antibodies, and analyzed by flow cytometry. Each bar represents data from 5–9 mice (error bars indicate SEM). *p < 0.05, **p < 0.01, ***p < 0.001. See also
Deconvoluting the Phenotype of motheaten Mice
496 Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc.
example, we saw enhanced activating phosphorylation of SFKs
and the MAPKs Erk 1 and 2 in response to integrin ligation, as
well as increased superoxide production by neutrophils plated
onto integrin ligands. Most importantly, neutrophil-specific
deletion of Syk, a critical mediator of integrin signals, prevented
hyperactive integrin signaling in Ptpn6fl/fS100a8-cre neutro-
phils, as well as the inflammatory phenotype, in these mice.
Similarly, deficiency of SFKs reversed the inflammatory pheno-
type in Ptpn6me-v/me-vmice. The trigger that initiates the inflam-
matory process in the skin is unclear, although it is interesting
that disease is localized to the paws. Perhaps inflammation is
initiated by small injuries, caused by walking or scratching,
that signal neutrophil recruitment. The hyperresponsiveness
of the Shp1-deficient neutrophils could then lead to an
increased local inflammatory response, causing a self-ampli-
fying loop of dermal inflammation. Consistent with this hypoth-
esis, we noticed that the onset of inflammation in mice housed
alone occurred later than in those housed with littermates (data
not shown). The more widespread skin inflammation present in
motheaten mice (compared with that seen in Ptpn6fl/fS100a8-
cre mice) might reflect the contribution of other Shp1-deficient
immune cells to the overall inflammatory phenotype.
It is clear, however, that amplified MyD88-mediated sig-
naling is not involved in initiating the inflammatory disease in
motheaten mice. If anything, the absence of MyD88 seemed to
impart a developmental defect on this strain, because we were
unable to generate any live-born Ptpn6me-v/me-vMyd88?/?or
Ptpn6fl/flMyd88fl/flVav1-cre animals. Ptpn6fl/flMyd88fl/flS100a8-
cre mice exhibited the same inflammatory disease seen in the
single-mutant Ptpn6fl/fS100a8-cre animals, demonstrating that
neutrophil-mediated inflammation was not driven by hyperacti-
vation of neutrophil-intrinsic TLR or IL-1b signaling through
MyD88. Our results differ from the conclusions of earlier work
using Ptpn6spin/spinmice, which were obtained by ENU-induced
Figure 6. Splenic DCs from Ptpn6fl/flItgax-cre Mice Show Exaggerated TLR Signaling
(A) BMDCs from mice of the indicated genotypes were stimulated with 100 ng/ml LPS for the indicated times, lysed and analyzed by SDS-PAGE followed by
immunoblotting with the indicated antibodies. Data shown are representative of at least five individual experiments.
(B) Splenocytes harvested from mice of the indicated genotypes were stimulated with 1 mg/ml LPS or 2 mg/ml CpG for the indicated times. Cells were then fixed
with formaldehyde, permeabilized with methanol, stained with fluorescently conjugated anti-phospho-Erk, and analyzed by flow cytometry. The histograms
represent the CD11chiDC gate.
(C) Splenocytes were harvested from mice of the indicated genotypes, treated with brefeldin A, and stimulated with 1 mg/ml LPS or 2 mg/ml CpG for 5 hr. Cells
were fixed and stained with fluorescently labeled anti-TNF-a and anti-CD11c, and analyzed by flow cytometry. Data in (B) and (C) are representative of three
independent experiments done on different cohorts of mice. See also Figure S5.
Deconvoluting the Phenotype of motheaten Mice
Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc. 497
mutagenesis of C57BL/6J mice and contain a Y208N amino acid
paw inflammation does not occur when these mice are crossed
with Myd88poc/pocmice (Croker et al., 2008), and inflammatory
disease in Ptpn6spin/spinmice has been attributed to hyperactive
IL-1b signaling in neutrophils (Croker et al., 2011). There are
several possible reasons for the apparent contradiction between
our conclusions and those of Croker and colleagues. First, we
specifically removed Shp1 from neutrophils using S100a8-cre-
directed deletion. Conceivably, indirect effects of other cells on
neutrophils could explain the earlier findings, because the
Ptpn6spin/spinmutation is present in all cells. Second, the
Ptpn6spin/spinmutation results in a Shp1 protein containing
a nonfunctional SH2 domain, whereas Ptpn6fl/flS100a8-cre
mice have a complete absence of Shp1. Finally, Croker et al.
used a point mutant of MyD88 that reduces signaling, whereas
we used mice that lacked MyD88 completely.
The fact that these studies produced such different results
might highlight the roles of individual domains of Shp1 in
immune cell function. Shp1 reportedly has phosphatase-
dependent and -independent functions (An et al., 2008), so
different mutants might have diverse effects on cell signaling.
Indeed, we showed previously that some Shp1 substrates are
hyperphosphorylated in cells from Ptpn6me-v/me-vmice, wherein
Shp1 is present but has reduced phosphatase activity, but hy-
pophosphorylated in Ptpn6me/mecells, which lack Shp1 expres-
sion (Timms et al., 1998). A point mutation affecting an SH2
domain but leaving the phosphatase domain intact might well
act differently than a mutation that leads to complete loss of
In contrast to Ptpn6fl/flS100a8-cre mice, Ptpn6fl/flItgax-cre
mice showed a dramatic expansion of myeloid cells and an
increase in serum cytokines. These alterations impacted the
WT lymphoid compartment, in which Shp1 continued to be ex-
pressed at WT amounts, leading to T and B cell activation and
plasma cell expansion. Ptpn6fl/+Itgax-cre mice also developed
autoimmune disease, consistent with previous observations
that Ptpn6me-v/+mice produce autoantibodies (Kuntz et al.,
1990). As in neutrophils, Shp1 deficiency in DCs did not cause
global activation of multiple signaling pathways; instead,
splenic DCs from Ptpn6fl/flItgax-cre mice were hyperresponsive
specifically to TLR ligands, including LPS and CpG. This
enhancement of TLR signaling is critical for autoimmunity,
because simultaneous deletion of Myd88 prevented disease
development in these mice. Autoimmune disease traditionally
has been attributed to altered B or T cell function, but our find-
ings indicate that dysregulation of DC-intrinsic TLR signaling
alone can drive disease. Similarly, DC-specific deletion of
A20, a negative regulator of NF-kB signaling, reportedly leads
to autoimmunity (Kool et al., 2011), although this finding is
controversial (Hammer et al., 2011). The ability of TLR signaling
to drive autoimmunity is consistent with the fact that human
autoimmune disease often is exacerbated by concurrent infec-
tions that could provide signals through TLRs (Chervonsky,
2010; Marshak-Rothstein, 2006). Our report demonstrates
that exaggerated TLR signaling in DCs alone can drive autoim-
mune disease development.
How Shp1 impacts TLR signaling remains unclear. Previous
studies have reported positive and negative effects via direct
or indirect mechanisms (An et al., 2008; Hardin et al., 2006;
Figure 7. Deletion of Myd88 in DCs Reverses the Exaggerated TLR Signaling in Ptpn6fl/flItgax-cre Mice
(A) Splenocytes were harvested from mice of the indicated genotypes, and then stimulated with or without 1 mg/ml LPS for 30 min. Cells were fixed with
formaldehyde, permeabilized with methanol, and stained with fluorescently conjugated anti-phospho-Erk, followed by flow cytometry. The histograms represent
(B) Splenocytes were harvested from mice with the indicated genotypes, then treated with brefeldin A and stimulated with 1 mg/ml LPS for 5 hr. Cells were fixed,
stained with fluorescently conjugated antibodies, including anti-TNF-a, and analyzed by flow cytometry. Dot plots show CD11chicells.
Data in (A) and (B) are representative of three independent experiments.
Deconvoluting the Phenotype of motheaten Mice
498 Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc.
Ramachandran et al., 2011; Rego et al., 2011; Zhou et al., 2010).
These studies are complicated, if not compromised, by cell-type
specificity and the variety of different Shp1 mutations used.
Furthermore, experiments exploring the effects of acute Ptpn6
deletion could highlight aberrant signaling pathways that can
be compensated for in cells with chronic Shp1 deficiency. For
from Ptpn6me-v/me-vmice produce WT amounts of TNF-a and
IL-6, but less IL-12p40 (Zhou et al., 2010), whereas the same
cells isolated from Ptpn6me/memice produce more TNF-a, but
less IL-6 (Rego et al., 2011). Peritoneal macrophages from
Ptpn6spin/spinmice show no change in TNF-a production in
response to TLR ligands, in contrast to the neutrophils from
these animals, which produce increased levels of TNF-a
(Croker et al., 2008; Croker et al., 2011). Furthermore, WT
BMDCs subjected to three independent methods of acute
Shp1 inhibition produce elevated IL-12p70, IL-1b, and IL-6, but
not TNF-a following LPS treatment (Ramachandran et al.,
2011). The authors of this study further noted that BMDCs
from Ptpn6me-v/me-vmice are less responsive to LPS, showing
that different results are observed depending on how Shp1 is
inactivated. Shp1 has been proposed to inhibit TLR signals in a
phosphatase-dependent manner by dephosphorylating IRAK4
(Ramachandran et al., 2011) or by inhibiting PI-3 kinase (Zhou
et al., 2010) and in a phosphatase-independent way by binding
to and inhibiting the kinase domain of IRAK1 (An et al., 2008).
These studies highlight that the precise mechanism by which
Shp1 modulates TLR responses remains confusing; neverthe-
less, our results clearly demonstrate that this regulation is phys-
iologically significant, especially in DCs.
phages (afforded by the Itgax-cre transgene) or both was not
sufficient to cause major cellular infiltration into the lung, even
though both of these cell types are found in the lungs of
motheaten mice. Presumably, pneumonitis requires Shp1 defi-
ciency in other cell types. Other lung diseases are dependent
on platelets (Looney et al., 2009), so their contribution to pulmo-
nary disease in the motheaten model merits future investigation.
Similarly, the contribution of mast cells should be considered,
given the partial rescue of the lung phenotype in motheaten
mice crossed onto a mast cell-deficient background such as
KitW-sh/W-shor KitW-v/W-v(Lorenz et al., 1996; Paulson et al.,
1996; Zhang et al., 2010). Previous work also has shown that
pulmonary inflammation in Ptpn6me-v/me-vmice is significantly
ameliorated by concomitant IL-13 or Stat6 deletion, but the
cell type involved is unknown (Oh et al., 2009).
Our results, combined with previous reports on the effects
of B cell- and T cell-specific Ptpn6 deletion, provide a much
improved understanding of the relative contributions of different
hematopoietic cells to the overall motheaten phenotype (Fowler
et al., 2010; Pao et al., 2007b). Future work will be required to
dissect the specific molecular interactions, including cytokine
networks, between these cells and their contribution to the
complex disease present in motheaten mice. Given the recent
observation of mutations in Ptpn6 in human neutrophilic derma-
toses that mimic the inflammatory disease seen in motheaten
mice (Nesterovitch et al., 2011b), and the emerging causal role
of DCs in autoimmune disease, understanding how Shp1
functions in different cell types should provide insights into
developing targeted therapies to treat human inflammatory and
Mice and Reagents
Details of mouse strains used can be found in Supplemental Experimental
Procedures. All mice were kept in a specific pathogen-free facility at the
University of California, San Francisco (UCSF), and cared for in accordance
with UCSF institutional guidelines.
Cell Preparation and Flow Cytometry
Single cell suspensions were prepared by homogenizing spleens between
two frosted microscope slides, followed by passage through a 70 mM cell
strainer. Cell numbers were determined by using a Nucleocounter (Chemo-
metec). Peripheral blood was collected in microtainer tubes with EDTA (BD
Biosciences), and CBCs were obtained by using a Hemavet 950 (Drew Scien-
tific). Bronchoalveolar lavage cells were obtained with five 1 ml flushes of the
lungs with ice-cold 5 mM EDTA in PBS. Peritoneal lavage cells were obtained
with a 10 ml flush of the peritoneum with ice cold 2% (vol/vol) FCS in PBS.
Red blood cells were removed from all cell suspensions by lysis with ACK
buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA pH 7.2–7.4). Bone-
marrow cell suspensions were obtained by flushing femurs and tibiae, lysing
red blood cells with a hypotonic NaCl solution, and passage through a 70 mM
cell strainer. Cells were resuspended in Hanks-buffered saline solution con-
taining 2% (vol/vol) fetal calf serum, 20 mM HEPES, and 1 mM EDTA and
maintained at 4?C. For flow cytometry, 1–2 3 106cells were stained. Nonspe-
cific binding was blocked by preincubation with 0.5 mg anti-CD16/32 (2.4G2,
UCSF Immunology Hybridoma Core) and 100 mg mouse IgG (Sigma).
For intracellular staining of Shp1, spleen or bone-marrow cells, stained for
ences), blocked with 2.4G2 and murine IgG as before, stained with anti-Shp1-
PE (Santa Cruz Biotechnology), washed with Perm/Wash (BD Biosciences),
and analyzed by flow cytometry.
For phosphoflow experiments, bone-marrow cells or splenocytes were
resuspended in 10% (vol/vol) FCS in RPMI at 2 3 106cells/ml and incu-
bated for 1 hr at 37?C, 5% CO2. Stimuli were added for the indicated times,
and then cells were fixed with 1.5% (vol/vol) methanol-free formaldehyde
(Thermo-Scientific) at RT for 15 min, followed by permeabilization in ice-
cold methanol for 15–30 min on ice or overnight at ?80?C. Fixed cells were
washed with 0.5% (wt/vol) BSA, 0.02% (wt/vol) NaAz in PBS, blocked with
2.4G2 and murine IgG as before and stained with the following antibodies in
addition to those listed in the Supplemental Experimental Procedures: anti-
phospho-Erk (Cell Signaling Technology), donkey anti-rabbit IgG-APC (Jack-
son Laboratories), anti-phospho Stat3-FITC, and anti-phospho Stat5-FITC
For intracellular cytokine staining, splenocytes were resuspended in 10%
(vol/vol) FCS in RPMI at 2 3 106cells/ml and stimulated with 1 mg/ml LPS or
2 mg/ml CpG for 5 hr at 37?C, 5% CO2in the presence of 10 mg/ml Brefeldin
A (eBiosciences). Fixed cells were washed and stained for surface markers
as above in the presence of Brefeldin A, then incubated in Fixation/Permeabi-
lization Concentrate diluted 1:4 with Diluent solution (eBiosciences). Cells
were blocked with 2.4G2 and murine IgG, stained with anti-TNF-a-FITC
(eBiosciences), washed with Permeabilization buffer (eBiosciences), and
analyzed by flow cytometry.
CD11chisplenic DCs were purified with a PE conjugated anti-CD11c
followed by anti-PE microbeads (Miltenyi Biotec). The positive fraction
was eluted using Miltenyi Biotec columns, stained as described above,
and sorted on a FACs Aria (Becton Dickinson) to >95% purity. CD11c+
splenic DCs were purified using the mouse CD11c+selection kit (StemCell
Tissues were fixed in 10% (vol/vol) formalin, embedded in paraffin and stained
with hematoxylin and eosin by the UCSF Pathology core. Paws were fixed in
10% (vol/vol) buffered formalin solution (Sigma) for 2 days, rinsed with tap
water, incubated in Cal-Ex (Fisher Scientific) for 2–3 days, and cut longitudi-
nally before being processed as above.
Deconvoluting the Phenotype of motheaten Mice
Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc. 499
Detection of Serum Anti-Nuclear Antibodies and Cytokines
Blood was collected in Z-gel microtubes (Sarstedt) and left to clot at RT for
30–45 min. Then, tubes were centrifuged at 6,000 3 g for 2 min to obtain
serum. Sera were diluted 1:40 and applied to Kallestad HEp-2 slides (Bio-
Rad). Anti-nuclear antibodies were detected using FITC conjugated goat
tion, Adobe Photoshop was used to determine mean pixel intensity in the
green channel. For anti-dsDNA Ig ELISA, 96-well flat-bottom polystyrene
plates (Microtest 351172, BD) were coated with 20 ng/well of linearized
pUC19 plasmid in 100 mM Tris-HCl. After overnight incubation at room
temperature, plates were blocked with PBS containing 2% (vol/vol) FCS and
0.05% (vol/vol) Tween 20 for 30 min. Sera, diluted 1:40–1:600, were added
to the plate and incubated for 2 hr at room temperature. The assays were
developed with horseradish-peroxidase-conjugated goat anti-mouse IgG
(1:3,000) or IgM (1:5,000) (Bethyl Laboratories). After addition of TMB Micro-
well Peroxidase Substrate System (KPL), reactions were terminated with
1 M phosphoric acid (Sigma-Aldrich), and the absorbance at 450 nm (A450)
was measured by using a Spectra Max Plus microplate reader (Molecular
Cytokineand chemokine concentrations
quantified using a Milliplex?MAP mouse cytokine and chemokine kit from
Millipore, according to the manufacturer’s instructions. The concentration of
TNF-a in BMDC culture supernatants was determined by ELISA (R&D
in mousesera were
Cellular Functional Assays and Biochemical Analysis
Neutrophils, purified from bone marrow as described previously (Mo ´csai
et al., 2002, 2006) or by using a five-step gradient (Siemsen et al., 2007),
were stimulated as described (Mo ´csai et al., 2002, 2006). Adhesion-depen-
dent respiratory burst was measured as reported previously (Mo ´csai et al.,
2002) or by using isoluminol-enhanced chemiluminescence (Dahlgren
et al., 2007). Bone-marrow-derived macrophages were differentiated
in vitro from mononuclear cells as described (Mo ´csai et al., 2006). BMDCs
were differentiated in vitro from mononuclear cells cultured for 11 days in
medium containing GM-CSF. Cells were serum-starved for 12 hr prior to
stimulation. Bone-marrow mononuclear cells were spin-infected with retro-
virus as described (Mo ´csai et al., 2006), followed by differentiation in medium
disease-free mice was plotted by using Kaplan-Meir survival analysis and
analyzed by using a log-rank (Mantel-Cox) test. Differences between two
groups were assessed by the unpaired t test; differences between three or
more groups were evaluated by ANOVA, followed by Bonferroni’s Multiple
Comparison post-test. Differences between observed and expected allele
when p % 0.05.
Supplemental Information includes five figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.doi.
We thank Yongmei Hu for help with animal husbandry and genotyping;
and Chrystelle Lamagna for help with FACS sorting. Supported by the US
National Institutes of Health (AI065495, AI068150 and AI078869 to C.A.L.,
RO1CA114945 and R37CA49152 to B.G.N.). B.G.N. is a Canada Research
Chair, Tier I, and is partially supported by the Ontario Ministry of Health and
Long Term Care and the Princess Margaret Hospital Foundation.
Received: May 4, 2012
Accepted: November 26, 2012
Published: March 21, 2013
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